- Email: [email protected]
204. Development of a Safe and Tumor-Targeting AAV Vector with a Strong Cancer-Specific Expression System
Cancer - Targeted Gene Therapy: Virotherapy where not evident in SH. Following i.c.i. of AdWT or CRAd-S-pk7, there appears to be a transient liver transaminitis without evidence of toxicity in these permissive models.
202. Intravenous Treatment with ThirdGeneration Oncolytic Herpes Simplex Viruses for Metastatic Renal Cell Carcinoma
Yuzuri Tsurumaki,1 Hiroshi Fukuhara,1 Tadaichi Kitamura,1 Yasushi Ino,2 Tomoki Todo.2,3 1 Urology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; 2Neuro-Oncology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; 3 Neurosurgery, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. Oncolytic herpes simplex viruses are promising therapeutic reagents for cancer. We developed a system for constructing armed oncolytic HSV-1 vectors that allowed insertion of desired transgenes into the deleted ICP6 locus of the triple-mutated HSV-1, G47∆. Using this system, we generated T-mfIL12 that expresses the fusion-type murine interleukin 12 (mIL-12). Culture media of Vero cells and RenCa (murine renal cell carcinoma) cells infected with T-mfIL12 at MOI=2 contained 20.8 and 40.7 ng/ml of mIL-12, respectively, 48h after infection. Human renal cell carcinoma cell lines, A498, ACHN, Caki-1, OS-RC-2 and RCC10RGB, and a murine cell line, RenCa, were used for further in vitro evaluation. All cell lines were susceptible to T-01, the control HSV-1 vector, at MOI=0.1, with the percentage of surviving cells ranging from 11 to 73% at day 3. T-01 and T-mfIL12 showed comparable cytopathic activities in vitro, which suggests that the insertion of the mIL-12 gene does not interfere with the replication capability of the parent virus. In athymic mice with subcutaneous human OS-RC-2 tumors, intraneoplastic inoculation with T-01 (4x 104, 2x 105 or 1x 106pfu) caused a dose dependent inhibition of the tumor growth. In BALB/c mice with subcutaneous murine RenCa tumors, both T-01 and T-mfIL12 showed significantly greater inhibition of the tumor growth compared with mock when inoculated intraneoplastically, with T-mfIL12 being significantly more efficacious than T-01. The efficacy of intravenous administration with the viruses was evaluated using the RenCa lung metastases model. BALB/c mice received intravenous injections of RenCa cells, and T-01 or T-mfIL12 (5x 105 or 5x 106 pfu) was administered into the tail vein on days 1, 3 and 5. Animals were either sacrificed at day 14 to count the number of lung metastases, or, in different sets of experiment, observed for survival. T-mfIL12 was significantly more efficacious than T-01 in reducing the number of lung metastases: At the dose of 5 x 106 pfu, the mean numbers of metastases were 326.3, 7.1 and 0.4 for mock, T-01 and T-mfIL12, respectively, and at the dose of 5 x 105 pfu, they were 204.8, 42.3 and 1.9, respectively. T-mfIL12 was also significantly more efficacious in prolonging the survival of animals than T-01 at both doses tested. When intravenous treatments started 9 days after the injection of RenCa cells, T-mfIL12, but not T-01, prolonged the survival compared with mock. In summary, thirdgeneration oncolytic HSV-1 vectors showed significant antitumor efficacy against renal cell carcinoma both in vitro and in vivo. Further, arming the oncolytic HSV-1 with IL-12 resulted in significant augmentation of the in vivo efficacy. Intravenous administration may be a useful means of delivery for the treatment of renal cell carcinoma, especially for lung metastases.
203. Targeting Carcinoembryonic Antigen (CEA) for Herpes Simplex Virus Infection
Hyunjung Baek,1,2 Jae-hong Kim,2 Masahide Kuroki,3 Willam F. Goins,4 Justus B. Cohen,4 Joseph C. Glorioso,4 Heechung Kwon.1 1 Laboratory of Molecular Oncology, Korea Institute of Radiological and Medical Sciences, Seoul, Korea; 2School of Life Sciences and Biotechnology, Korea University, Seoul, Korea; 3 Molecular Oncology and First Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka, Japan; 4 Department of Microbiology and Molecular Genetics, Biomedical Science Tower, University of Pittsburgh, Pittsburgh, PA.
Herpes simplex virus infects and spreads in epithelial cells, neurons, and cells of the immune system. The broad host range of HSV-1 is an obstacle to the development of this virus into a gene transfer vector for therapeutic applications. We are studying approaches to redirect the natural tropism of the virus. HSV-1 infection depends on the interaction of one of its essential envelope glycoproteins, gD, with a cognate cellular receptor including nectin-1/HveC, HVEM/HveA, and 3-O-sulfated heparan sulfate. We previously reported that the soluble V domain of nectin-1/HveC has the ability to mediate HSV infection of HSV-resistant CHO-K1 cells. We now show that this property is not limited to nectin-1. A soluble portion of the HVEM/HveA ectodomain, composed of the first two cysteine-rich pseudorepeat (CRP) domains (sHveA102), can efficiently mediate HSV infection of gD-receptor deficient CHO cells. As measured by viral reporter gene expression, sHveA102 caused infection of greater than 80% of the cells at an MOI of 3. To investigate whether the tumor antigen CEA can be targeted to mediate HSV infection, we constructed a recombinant adapter, F39-HveA, composed of a single chain anti-CEA antibody linked to sHveA102. Preincubation of HSV-1 with F39-HveA and subsequent infection of CHO cells expressing CEA resulted in infection of these HSV-resistant cells. F39-HveA-mediated infection was blocked by soluble gD ectodomain as well as by anti-CEA monoclonal antibody, indicating that virus entry was dependent on both viral gD and CEA. Furthermore, F39-HveA increased HSV infection of CEA-expressing human cancer cells when virus entry through nectin-1 was blocked by nectin-1 antibody or full-length soluble nectin-1 ectodomain. These results demonstrate that a targeted tumor antigen can function as an HSV-1 entry receptor. Current studies are aimed at determining whether this approach in combination with certain host-rangerestricted mutant viruses can provide sufficient tumor cell selectivity for further development as an oncolytic therapy.
204. Development of a Safe and TumorTargeting AAV Vector with a Strong CancerSpecific Expression System
Keerang Park,1 Wongi Seol,2 Young-Hwa Cho,1 Hye Jin Yun,2 Sung-Ha Cho,1 Heejong Kim,1 Bong-Su Kang,1 Tae Sup Lee,3 In Ho Song,3 Gi Jeong Cheon,3 Sunjoo Jeong,4 Heuiran Lee.5 1 Juseong Gene Therapy R&D Center, Department of Biotechnology, Juseong College, Chungbuk, Republic of Korea; 2 Graduate Program in Neuroscience, Institute for Brain Science and Technology (IBST), Inje University, Pusan, Republic of Korea; 3Division of Nuclear Medicine and RI, Korea Institute of Radiological and Medical Sciences (KIRAMS), Seoul, Republic of Korea; 4Department of Molecular Biology, BK21 Graduate Program for RNA Biology, Institute of Nanosensor and Biotechnology, Dankook University, Gyeonggi, Republic of Korea; 5Department of Microbiology, Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul, Republic of Korea. To establish gene therapeutic medicines for cancers, a safe and tumor-targeting gene delivery vector with a strong cancer-specific expression system needs to be developed. For several years, we
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Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Cancer - Targeted Gene Therapy: Virotherapy have investigated how efficiently a therapeutic gene can be delivered specifically to cancers via AAV-mediated delivery systems. Our previous results demonstrated that AAV serotype 2(AAV2) showed the highest transduction efficiency into human cancer cell lines and AAV5 was the second best. To determine in vivo cancer tropism of AAV vectors, we used a Xenogen IVIS 200 bioluminescent imaging (BLI) system. After intra-venous(IV), intra-peritoneal(IP) or intratumoral(IT) administration of the HeLa-implanted nude mice with either AAV2 or AAV5 vectors containing a luciferase reporter gene, the gene expressions were monitored. The gene expressions by rAAV2-CMV-Luc vectors were detectable only at tumors of the IT-injected mice on day 2. In contrast, the gene expressions in the IP-injected mice with AAV2 were seen in most of tissues including the strongest expression at tumors, whereas those in the IV-injected mice with AAV2 vectors were mainly localized to tumors and livers. Those gene expressions in all the treated mice were getting stronger and persistent to day 23 after injection. On the other hand, the gene expressions in the treated mice with rAAV5-CMV-Luc vectors were slower and weaker than those with AAV2, but the expression profiles in the IV- and IT-injected mice with AAV5 were similar to those with AAV2. However, the expression profiles in the IP-injected mice with AAV5 were quite different from those with AAV2. We then examined putative cancer-specific promoters of PRC1, RRM2 and BIRC5 genes in the nude mice implanted with HeLa, MDA-MB-231 or A549 using a BLI system. The gene expressions in the IV- or ITtreated mice with rAAV2-PRC1-Luc were stronger and faster than those with rAAV2-CMV-Luc. Although the expressions in both AAV vectors-injected mice became stronger and persistent to day 20, the gene expressions in the rAAV2-PRC1-Luc-injected mice were mainly localized to tumors and livers as those in the rAAV2-CMV-Luc-treated mice were seen. Taken altogether, our present study suggested that both of AAV2 and AAV5 vectors can be further developed as more efficient cancer therapeutic vectors by modifications of PRC1 putative cancer-specific promoter.
205. Lentiviral Pseudotyped Vectors Specifically and Efficiently Transduce Invasive Human Glioblastoma Xenografts
In contrast, retroviral LMCV-GP vectors were much less efficient in transducing high generation spheroids and did not transduce tumor cells of low generation spheroids (figure1). In vivo, both lentiviral vectors specifically and efficiently transduced glioblastoma xenografts in solid and even in invasive areas (figure2A-D).
Hrvoje Miletic,1 Peter Huszthy,1 Tsanan Giroglou,2 Rolf Bjerkvig,1 Dorothee von Laer.2 1 Department of Biomedicine, University of Bergen, Bergen, Norway; 2Georg-Speyer-Haus, Frankfurt am Main, Germany.
Lentiviral vectors pseudotyped with glycoproteins of the Lymphocytic choriomeningitis virus (LCMV-GP) are promising candidates for gene therapy of malignant glioma, as they show efficient transduction and therapy of rat glioma cells in vitro and in vivo (Miletic et al., Human Gene Ther. 2004 and Clinical Cancer Res. 2007). Here, we investigated the transduction efficacy of LCMV-GP and Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors versus Murine Leukemia Virus (MLV) derived retroviral LCMV-GP pseudotyped vectors for cultured spheroids from human glioblastoma and in a recently established glioma animal model. This model is derived from human glioblastoma spheroids and shows all hallmarks of tumor growth in patients with invasion, angiogenesis and necrosis (Sakariassen et al., PNAS 2006). In vitro, both lentiviral LCMV-GP and VSV-G pseudotyped vectors transduced spheroids directly from patients (low generation) and after serial passaging in nude rats (high generation)(figure1).
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
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202. Intravenous Treatment with ThirdGeneration Oncolytic Herpes Simplex Viruses for Metastatic Renal Cell Carcinoma
Yuzuri Tsurumaki,1 Hiroshi Fukuhara,1 Tadaichi Kitamura,1 Yasushi Ino,2 Tomoki Todo.2,3 1 Urology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; 2Neuro-Oncology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; 3 Neurosurgery, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. Oncolytic herpes simplex viruses are promising therapeutic reagents for cancer. We developed a system for constructing armed oncolytic HSV-1 vectors that allowed insertion of desired transgenes into the deleted ICP6 locus of the triple-mutated HSV-1, G47∆. Using this system, we generated T-mfIL12 that expresses the fusion-type murine interleukin 12 (mIL-12). Culture media of Vero cells and RenCa (murine renal cell carcinoma) cells infected with T-mfIL12 at MOI=2 contained 20.8 and 40.7 ng/ml of mIL-12, respectively, 48h after infection. Human renal cell carcinoma cell lines, A498, ACHN, Caki-1, OS-RC-2 and RCC10RGB, and a murine cell line, RenCa, were used for further in vitro evaluation. All cell lines were susceptible to T-01, the control HSV-1 vector, at MOI=0.1, with the percentage of surviving cells ranging from 11 to 73% at day 3. T-01 and T-mfIL12 showed comparable cytopathic activities in vitro, which suggests that the insertion of the mIL-12 gene does not interfere with the replication capability of the parent virus. In athymic mice with subcutaneous human OS-RC-2 tumors, intraneoplastic inoculation with T-01 (4x 104, 2x 105 or 1x 106pfu) caused a dose dependent inhibition of the tumor growth. In BALB/c mice with subcutaneous murine RenCa tumors, both T-01 and T-mfIL12 showed significantly greater inhibition of the tumor growth compared with mock when inoculated intraneoplastically, with T-mfIL12 being significantly more efficacious than T-01. The efficacy of intravenous administration with the viruses was evaluated using the RenCa lung metastases model. BALB/c mice received intravenous injections of RenCa cells, and T-01 or T-mfIL12 (5x 105 or 5x 106 pfu) was administered into the tail vein on days 1, 3 and 5. Animals were either sacrificed at day 14 to count the number of lung metastases, or, in different sets of experiment, observed for survival. T-mfIL12 was significantly more efficacious than T-01 in reducing the number of lung metastases: At the dose of 5 x 106 pfu, the mean numbers of metastases were 326.3, 7.1 and 0.4 for mock, T-01 and T-mfIL12, respectively, and at the dose of 5 x 105 pfu, they were 204.8, 42.3 and 1.9, respectively. T-mfIL12 was also significantly more efficacious in prolonging the survival of animals than T-01 at both doses tested. When intravenous treatments started 9 days after the injection of RenCa cells, T-mfIL12, but not T-01, prolonged the survival compared with mock. In summary, thirdgeneration oncolytic HSV-1 vectors showed significant antitumor efficacy against renal cell carcinoma both in vitro and in vivo. Further, arming the oncolytic HSV-1 with IL-12 resulted in significant augmentation of the in vivo efficacy. Intravenous administration may be a useful means of delivery for the treatment of renal cell carcinoma, especially for lung metastases.
203. Targeting Carcinoembryonic Antigen (CEA) for Herpes Simplex Virus Infection
Hyunjung Baek,1,2 Jae-hong Kim,2 Masahide Kuroki,3 Willam F. Goins,4 Justus B. Cohen,4 Joseph C. Glorioso,4 Heechung Kwon.1 1 Laboratory of Molecular Oncology, Korea Institute of Radiological and Medical Sciences, Seoul, Korea; 2School of Life Sciences and Biotechnology, Korea University, Seoul, Korea; 3 Molecular Oncology and First Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka, Japan; 4 Department of Microbiology and Molecular Genetics, Biomedical Science Tower, University of Pittsburgh, Pittsburgh, PA.
Herpes simplex virus infects and spreads in epithelial cells, neurons, and cells of the immune system. The broad host range of HSV-1 is an obstacle to the development of this virus into a gene transfer vector for therapeutic applications. We are studying approaches to redirect the natural tropism of the virus. HSV-1 infection depends on the interaction of one of its essential envelope glycoproteins, gD, with a cognate cellular receptor including nectin-1/HveC, HVEM/HveA, and 3-O-sulfated heparan sulfate. We previously reported that the soluble V domain of nectin-1/HveC has the ability to mediate HSV infection of HSV-resistant CHO-K1 cells. We now show that this property is not limited to nectin-1. A soluble portion of the HVEM/HveA ectodomain, composed of the first two cysteine-rich pseudorepeat (CRP) domains (sHveA102), can efficiently mediate HSV infection of gD-receptor deficient CHO cells. As measured by viral reporter gene expression, sHveA102 caused infection of greater than 80% of the cells at an MOI of 3. To investigate whether the tumor antigen CEA can be targeted to mediate HSV infection, we constructed a recombinant adapter, F39-HveA, composed of a single chain anti-CEA antibody linked to sHveA102. Preincubation of HSV-1 with F39-HveA and subsequent infection of CHO cells expressing CEA resulted in infection of these HSV-resistant cells. F39-HveA-mediated infection was blocked by soluble gD ectodomain as well as by anti-CEA monoclonal antibody, indicating that virus entry was dependent on both viral gD and CEA. Furthermore, F39-HveA increased HSV infection of CEA-expressing human cancer cells when virus entry through nectin-1 was blocked by nectin-1 antibody or full-length soluble nectin-1 ectodomain. These results demonstrate that a targeted tumor antigen can function as an HSV-1 entry receptor. Current studies are aimed at determining whether this approach in combination with certain host-rangerestricted mutant viruses can provide sufficient tumor cell selectivity for further development as an oncolytic therapy.
204. Development of a Safe and TumorTargeting AAV Vector with a Strong CancerSpecific Expression System
Keerang Park,1 Wongi Seol,2 Young-Hwa Cho,1 Hye Jin Yun,2 Sung-Ha Cho,1 Heejong Kim,1 Bong-Su Kang,1 Tae Sup Lee,3 In Ho Song,3 Gi Jeong Cheon,3 Sunjoo Jeong,4 Heuiran Lee.5 1 Juseong Gene Therapy R&D Center, Department of Biotechnology, Juseong College, Chungbuk, Republic of Korea; 2 Graduate Program in Neuroscience, Institute for Brain Science and Technology (IBST), Inje University, Pusan, Republic of Korea; 3Division of Nuclear Medicine and RI, Korea Institute of Radiological and Medical Sciences (KIRAMS), Seoul, Republic of Korea; 4Department of Molecular Biology, BK21 Graduate Program for RNA Biology, Institute of Nanosensor and Biotechnology, Dankook University, Gyeonggi, Republic of Korea; 5Department of Microbiology, Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul, Republic of Korea. To establish gene therapeutic medicines for cancers, a safe and tumor-targeting gene delivery vector with a strong cancer-specific expression system needs to be developed. For several years, we
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Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Cancer - Targeted Gene Therapy: Virotherapy have investigated how efficiently a therapeutic gene can be delivered specifically to cancers via AAV-mediated delivery systems. Our previous results demonstrated that AAV serotype 2(AAV2) showed the highest transduction efficiency into human cancer cell lines and AAV5 was the second best. To determine in vivo cancer tropism of AAV vectors, we used a Xenogen IVIS 200 bioluminescent imaging (BLI) system. After intra-venous(IV), intra-peritoneal(IP) or intratumoral(IT) administration of the HeLa-implanted nude mice with either AAV2 or AAV5 vectors containing a luciferase reporter gene, the gene expressions were monitored. The gene expressions by rAAV2-CMV-Luc vectors were detectable only at tumors of the IT-injected mice on day 2. In contrast, the gene expressions in the IP-injected mice with AAV2 were seen in most of tissues including the strongest expression at tumors, whereas those in the IV-injected mice with AAV2 vectors were mainly localized to tumors and livers. Those gene expressions in all the treated mice were getting stronger and persistent to day 23 after injection. On the other hand, the gene expressions in the treated mice with rAAV5-CMV-Luc vectors were slower and weaker than those with AAV2, but the expression profiles in the IV- and IT-injected mice with AAV5 were similar to those with AAV2. However, the expression profiles in the IP-injected mice with AAV5 were quite different from those with AAV2. We then examined putative cancer-specific promoters of PRC1, RRM2 and BIRC5 genes in the nude mice implanted with HeLa, MDA-MB-231 or A549 using a BLI system. The gene expressions in the IV- or ITtreated mice with rAAV2-PRC1-Luc were stronger and faster than those with rAAV2-CMV-Luc. Although the expressions in both AAV vectors-injected mice became stronger and persistent to day 20, the gene expressions in the rAAV2-PRC1-Luc-injected mice were mainly localized to tumors and livers as those in the rAAV2-CMV-Luc-treated mice were seen. Taken altogether, our present study suggested that both of AAV2 and AAV5 vectors can be further developed as more efficient cancer therapeutic vectors by modifications of PRC1 putative cancer-specific promoter.
205. Lentiviral Pseudotyped Vectors Specifically and Efficiently Transduce Invasive Human Glioblastoma Xenografts
In contrast, retroviral LMCV-GP vectors were much less efficient in transducing high generation spheroids and did not transduce tumor cells of low generation spheroids (figure1). In vivo, both lentiviral vectors specifically and efficiently transduced glioblastoma xenografts in solid and even in invasive areas (figure2A-D).
Hrvoje Miletic,1 Peter Huszthy,1 Tsanan Giroglou,2 Rolf Bjerkvig,1 Dorothee von Laer.2 1 Department of Biomedicine, University of Bergen, Bergen, Norway; 2Georg-Speyer-Haus, Frankfurt am Main, Germany.
Lentiviral vectors pseudotyped with glycoproteins of the Lymphocytic choriomeningitis virus (LCMV-GP) are promising candidates for gene therapy of malignant glioma, as they show efficient transduction and therapy of rat glioma cells in vitro and in vivo (Miletic et al., Human Gene Ther. 2004 and Clinical Cancer Res. 2007). Here, we investigated the transduction efficacy of LCMV-GP and Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors versus Murine Leukemia Virus (MLV) derived retroviral LCMV-GP pseudotyped vectors for cultured spheroids from human glioblastoma and in a recently established glioma animal model. This model is derived from human glioblastoma spheroids and shows all hallmarks of tumor growth in patients with invasion, angiogenesis and necrosis (Sakariassen et al., PNAS 2006). In vitro, both lentiviral LCMV-GP and VSV-G pseudotyped vectors transduced spheroids directly from patients (low generation) and after serial passaging in nude rats (high generation)(figure1).
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
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